Techniques are known for the analysis of compound such as drugs, and drug metabolites, such as are employed in screening for drugs of abuse in urine samples. The suspect chemical compounds are isolated by specific or generic counter-current extraction methods according to chemical class, and then analyzed. The analysis is made generally by such techniques as thin layer chromatography, immunoassay, gas chromatography using nitrogen/phosphorous or election capture detection, high pressure liquid chromatography (HPLC) using fluoresense, ultra violet or electrochemical detection, and the like. It should be expected that a compound such as a drug, or drug residue, extracted from a biological specimen, or tissue could be analyzed in similar manner if extracts of the biological specimens, or tissues, of adequately high purity could be prepared.
The preparation however of a high purity extract from, e.g., tissues presents a much more formidable problem than the manipulation of a urine matrix. It is well known that interfering substances abound in tissues, and that these substances must be eliminated from the extract before analysis is possible. For example, in thin layer chromatography, interfering substances must be absent, and lipid concentration must be reduced to levels which will prevent streaking of a sample. For radioimmunoassay analysis, e.g., isolation of the compounds of interest from the tissue matrix is required, as well as the removal of endogenous substances which produce false positive results. For gas chromatography and high pressure liquid chromatography there are a number of compounds that must be eliminated because they produce interfering peaks which make it difficult, or impossible, to detect trace levels of drugs and their metabolites.
Classical methods for tissue extraction involve homogenation of the tissues in an aqueous medium, multiple extractions and centrifuging for removal of debris, precipitation of proteins by adjustment of pH with acids, or bases, or by the addition of salts. Classical methods for the isolation of a compound such as a drug, or drug metabolite, from a matrix thus generally requires (1) mincing and/or mechanical homogenization of the tissue in an aqueous solvent; (2) addition of acids, bases, or salts to precipitate protein and remove debris; (3) centrifugations; (4) transfer of the supernatant and adjustment of pH; (5) counter-current extraction of the sample, often leading to intractable emulsions; and (6) back-extraction of the sample to assist in purification of the sample. Often also, homogenization in and repeated extraction of tissues by organic solvents are required, these generating large volumes of solvents which must be evaporated; and usually back-extracted. The necessity for multiple counter-current extractions of the isolated supernatant requires considerable time, and can adversely affect the accuracy of the analysis. Unfortunately such manipulations are burdensome per se, and invariably lead to emulsion formation and the necessity of further slow manipulations of the samples.
Solid phase extraction, SPE, technology has been used extensively over the past decade, but this technology has provided only a partial solution to the problem. With this technique an SPE column is packed with various materials; usually polymer-bound beads of various sizes. A supernatant of a homogenized, manipulated specimen of a tissue suspected to contain a compound, drug, or drug residue, is added to the SPE column, and this is followed by elution with a solvent, or solvents, to isolate a specific compound, drug, or class of drugs. The SPE process eliminates emulsion formation and lessens the volumes of solvent required to isolate a given drug, or compound, as contrasted with classical methods. Prior to passage of the sample through the SPE column however, homogenation of the tissue and manipulations of the specimen to remove cell debris is required. Often also it is required to remove the proteins and lipids. The removal of the cell debris, and as well the proteins and lipids, prevents plugging of the column and overloading the polymer phase. Accordingly, after homogenizing the tissue the homogenized specimen is pelletized by centrifugation, and the proteins precipitated. Thus, in this procedure most of the classical manipulations are still required, and it is necessary that the homogenized tissue specimen be pelletized in an aqueous medium prior to extraction. The necessity of having to pelletize the specimen is particularly disadvantageous in that entrained or electrostatically compounds, drugs, or drug residues, are sometimes lost. Moreover less than the whole of the tissue specimen is extracted in the SPE column. For these reasons, inter alia, the isolation of compounds such as drugs, and drug residues, from the matrices of biological specimens, or tissue matrices, via known solid phase extraction techniques have left much yet to be desired.